1. Department of Biochemistry and Molecular Biology, Medical College of Jinan University, Guangzhou 510632, China
2. Department of Hematology, Guangdong Second Provincial General Hospital, Jinan university, Guangzhou 510317, China
3. Guangdong Engineering Technology Research Center of Drug Development for Small Nucleic Acids, Guangzhou, China
4. Guangzhou Antisense Biopharmaceutical Technology Co., Ltd., Guangzhou 510632, China
5. Center Lab of Longhua Branch and Department of Infectious Disease, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University), Shenzhen, Guangdong 518020, China
6. Department of pathology (Longhua Branch), Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University), Shenzhen, Guangdong 518020, China
7. College of Pharmacy, Shenzhen Technology University, Shenzhen 518118, China
8. Department of Hematology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China
#Zhao Yin, Rui Su, Lanlan Ge, and Xiuyuan Wang contributed equally to this work.
BCR-ABL oncogene-mediated Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia (CML) is suggested to originate from leukemic stem cells (LSCs); however, factors regulating self-renewal of LSC and normal hematopoietic stem cells (HSCs) are largely unclear. Here, we show that RalA, a small GTPase in the Ras downstream signaling pathway, has a critical effect on regulating the self-renewal of LSCs and HSCs. A RalA knock-in mouse model (RalARosa26-Tg/+) was initially constructed on the basis of the Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR/Cas9) assay to analyze normal hematopoietic differentiation frequency using single-cell resolution and flow cytometry. RalA overexpression promoted cell cycle progression and increased the frequency of granulocyte-monocyte progenitors (GMPs), HSCs and multipotent progenitors (MPPs). The uniform manifold approximation and projection (UMAP) plot revealed heterogeneities in HSCs and progenitor cells (HSPCs) and identified the subclusters of HSCs and GMPs with a distinct molecular signature. RalA also promoted BCR-ABL-induced leukemogenesis and self-renewal of primary LSCs and shortened the survival of leukemic mice. RalA knockdown prolonged survival and promoted sensitivity to imatinib in a patient-derived tumor xenograft model. Immunoprecipitation plus single-cell RNA sequencing of the GMP population confirmed that RalA induced this effect by interacting with RAC1. RAC1 inhibition by azathioprine effectively reduced the self-renewal, colony formation ability of LSCs and prolonged the survival in BCR-ABL1-driven RalA overexpression CML mice. Collectively, RalA was detected to be a vital factor that regulates the abilities of HSCs and LSCs, thus facilitating BCR-ABL-triggered leukemia in mice. RalA inhibition serves as the therapeutic approach to eradicate LSCs in CML.
Keywords: RalA/single-cell resolution, leukemia stem cells, normal hematopoietic stem cells, imatinib resistance, mouse model with gene editing